1. Field of the Invention
The present invention relates generally to methods for synthesizing heat shock protein-peptide complexes.
2. Description of the Prior Art
Heat shock proteins (HSPs) are associated in cells with a broad spectrum of peptides, polypeptides, denatured proteins and antigens with which they form complexes. These complexes form as part of the cell's normal protein manufacturing process as described in Beckmann et al., "Interaction of Hsp70 with newly synthesized proteins: implications for protein folding and assembly" in Science (1990), 248: 850-4. HSP-peptide complexes may also be formed as part of the transport of peptides to the MHC class I molecules on the cell's surface as described in Srivastava et al., "Heat shock proteins transfer peptides during antigen processing" in Immunogenetics (1994), 39: 93-98. When purified from autologous tumors, such HSP-peptide complexes have been useful in vaccines against cancers and infectious diseases as described in Srivastava et al., "Heat shock protein-peptide complexes in cancer immunotherapy" in Current Opinion in Immunology (1994), 6: 728-732; and Srivastava, "Peptide-Binding Heat Shock Proteins in the Endoplasmic Reticulum" in Advances in Cancer Research (1993), 62: 153-177. Vaccination with antigenic peptides alone can elicit an immune response but peptides bound to HSPs appear to elicit a much more efficient response. In this regard, Srivastava has estimated in Srivastava, "Heat shock proteins in immune responses to cancer: the fourth paradigm" in Experientia (1994), 50: 1054-1060, and that 10 .mu.g of gp96 isolated from tumor cells may be complexed with approximately 10 pg of peptide representing a tumor-associated mutation. While 10 pg of peptide cannot be used to successfully vaccinate an animal against a subsequent tumor challenge, 10 pg of peptide complexed with gp96 is enough to elicit protection.
Several types of cancer vaccines that are currently being investigated, including heat shock protein vaccines, have been described in Old, "Immunotherapy for cancer" in Scientific American (1996), 275: 142. These potential vaccines have included: inactivated cancer cells and their extracts which can jump-start the immune system (particularly cancer cells engineered to secrete cytokines, such as IL-2 or GM-CSF, similarly heighten anti-tumor immunity and cells designed to express co-stimulatory molecules, such as B-1, enhanced the ability of T-cells to recognize tumor cells); tumor peptides, fragments of tumor proteins recognized by T-cells, which can be injected alone or with immune-boosting adjuvants; injected tumor proteins which are taken up by antigen-presenting cells and break them down into a range of peptide fragments recognized by T-cells; dendritic cells, antigen-presenting cells which are isolated from the blood, exposed to tumor peptides or engineered to produce tumor proteins and then reinjected; gangliosides, which humans can produce antibodies against and which are found on the surface of tumor cells; viral and bacterial vectors, genes coding for tumor antigens which are incorporated into viral or bacterial genomes, that, when injected, draw immunity against themselves and encoded antigens; and DNA and RNA coding for tumor antigens which prompt normal cells to begin producing these antigens.
The heat shock protein vaccines consist of HSP-peptide complexes purified from tumor cells containing a mixture of peptides as described by Udono and Srivastava in "Heat shock protein 70-associated peptides elicit specific cancer immunity" in Journal of Experimental Medicine (1993), 178: 1391-6. Most of the peptides complexed with HSPs and isolated from tumor cells are presumably portions of normal cellular proteins. Therefore, the HSP-peptide complexes need to be purified at fairly high levels in order to obtain HSPs complexed with peptide portions of mutated oncogenes. Because many of the purified peptides are portions of normal cellular proteins, vaccination with these purified complexes may incur the risk of auto-immune disorders.
Peptide vaccination experiments use peptide sequences of known tumor mutations such as described in Gjertsen et al., "Vaccination with mutant ras peptides and induction of T-cell responsiveness in pancreatic carcinoma patients carrying the corresponding RAS mutation" in Lancet (1995), 346: 1399-1400. Peptide vaccination experiments are also being tried with viral diseases such as described in Kelleher et al., "Safety and immunogenicity of UB1 HIV-IMN octameric V3 peptide vaccine administered by subcutaneous injection" in AIDS Research & Human Retroviruses (1997), 13: 29-32 are currently receiving much attention.
HSP-peptide complexes have been purified from autologous tumors and used for vaccination against the same tumor. It has been anticipated that material purified in this way can also be used for vaccination against human tumors in Srivastava and Udono, "Heat shock protein-peptide complexes in cancer immunotherapy" in Current Opinion in Immunology (1994), 6: 728-32. The use of these purified complexes in treating patient tumors would require the isolation of tumor material from each individual patient. This is not practical or even possible for many types of tumors. Many types of cancer, however, have well-defined mutations in specific genes. The use of de novo HSP-peptide complexes allows the use of HPS-peptide vaccines without the need for removing a portion of the tumor. The de novo complexes also allow prophylactic vaccination against a variety of diseases using peptides representing known oncogenic mutations or peptides representing portions of known viral proteins.
Bacterial hsp70-peptide complexes, created by incubating purified HSPs together with a synthetic peptide solution have also been used to stimulate peptide specific T-cell responses (See Roman et al., "Synthetic peptides non-covalently bound to bacterial hsp70 elicit peptide specific T-cell responses in vivo," Immunology, (1996), 88: 487-492, and Roman et al., "Delayed-type hypersensitivity elicited by synthetic peptides complexed with mycobacterial tuberculosis hsp70 ," Immunology, (1997), 90: 52-56).
In general, previous heat shock protein purifications have had two goals. The first goal has been to obtain pure HSPs without any contaminating peptides or proteins as described in Welch et al., "Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides" in Molecular and Cellular Biology (1985), 5: 1229-1237 and in Nandan et al., "A rapid single-step purification method for immunogenic members of the hsp family: validation and application" in Journal of Immunological Methods (1994), 176: 255-263. The second goal has been to purify the intracellular HSP-peptide complexes as described in Udono et al., "Heat shock protein 70-associated peptides elicit specific cancer immunity" in Journal of Experimental Medicine (1993), 178: 1391-1396.
However, the fact that previously described methods only produce HSPs already complexed with intracellular peptides or stripped of their associated peptides is a serious deficiency. Also, there is no known simple way of synthesizing purified HSP-peptide complexes.